Focus: DNA Purification

Isolation of Genomic DNA from Bacterial Cells using the Wizard^® SV Genomic DNA Purification System

By Ryan McDonald, M.Sc., and Olga Hill, MLT.
Molecular Diagnostics, Provincial Laboratory, Saskatchewan Health, Regina, SK, Canada

Introduction

With the emergence of antimicrobial-resistant organisms and the increased prevalence of food-borne human pathogens, surveillance of these bacteria has become an important interest to public health. For example, methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infections in health care facilities in many countries. Enteric bacteria such as Salmonella and Escherichia coli pose a threat to public health as causes of food-associated outbreaks. Molecular-based microbial subtyping methods have been refined over the past few years to aid in epidemiological investigation of outbreaks. One method, fluorescence-based Amplified Fragment Length Polymorphism (fbAFLP) DNA fingerprinting, has become a valuable technique for characterizing bacterial strains (1–3). For a clinical laboratory to be able to respond to increased numbers of outbreaks and isolates, methods such as fbAFLP must be adapted to handle high volumes by increasing throughput while decreasing turnaround-time.  fbAFLP requires a good yield of high quality genomic DNA from each isolate.  Conventional methods are labour-intensive and time-consuming, often requiring intricate manipulations and the use of hazardous organic chemicals. This results in a bottleneck in the fbAFLP procedure that limits the overall throughput and turnaround time for communication of results to the appropriate health care personnel. Introduction of an alternative DNA purification system must be considered to address these concerns.

Here we describe an application of the Wizard^® SV Genomic DNA Purification System for the purification of genomic DNA from Gram-positive and Gram-negative human pathogens including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Listeria monocytogenes, Salmonella Typhimurium, Salmonella Heidelberg and Escherichia coli O157:H7. The genomic DNA is isolated from overnight broth cultures and yields high quality DNA that serves as a good template for restriction/amplification analyses such as fbAFLP.

Methods and Results

Isolation of Genomic DNA from Gram-positive Bacteria
Isolation of Genomic DNA from Gram-negative Bacteria
Analysis of Purified Genomic DNA

Isolation of Genomic DNA from Gram-positive Bacteria

Materials to Be Supplied By the User

• Wizard^® SV Genomic DNA Purification System (Cat.#A2360, A2361)
• Lysozyme (1g) (Sigma Cat.# L7651)
• Lysostaphin (1g) (Sigma Cat.# 7386)
• 95% ethanol
• 80°C heat block or water bath
• 65°C heat block or water bath
• 37°C heat block or water bath
• 1.5ml microcentrifuge tubes
• microcentrifuge
• DNA Rehydration Solution (optional) (Cat.# A7963)

Preparation of Reagents

Prepare the Wizard^® SV Genomic DNA System components as directed in the Wizard^® SV Genomic DNA Purification System Technical Bulletin #TB302.

Lysozyme Working Solution: Completely dissolve the lysozyme powder by adding 20ml nuclease-free water to a 1g vial of lyzozyme and mix. Dispense into aliquots and store at –20°C. Avoid multiple freeze-thaw cycles. Lysozyme Working Solution is stable for 1 year at –20°C.

Lysostaphin Working Solution: Completely dissolve the lysostaphin powder by adding 500μl of nuclease-free water to a 5mg vial of lysostaphin and mix. Dispense into aliquots and store at –20°C. Avoid multiple freeze-thaw cycles. The Lysostaphin Working Solution is stable for 6 months at –20°C. 

Enzymatic Lysis Solution: For each extraction, combine 376μl of 50mM EDTA, 20μl of Lysozyme Working Solution and 4μl of Lysostaphin Working Solution.

Nuclei Lysis/RNase Solution: For each extraction, combine 500μl of Nuclei Lysis Solution and 7μl of RNase Solution (both are provided with the Wizard^® SV Genomic System).

Genomic DNA Purification Procedure: Gram-positive Bacteria

1. Pellet 0.5ml of overnight bacterial broth culture by centrifugation (21,000 × g for 2 minutes). Discard supernatant.
   
   
2. Resuspend the bacterial pellet in 400μl of freshly prepared Enzymatic Lysis Solution and incubate at 37°C for 60 minutes. At this time, place the DNA Rehydration Solution (or nuclease-free water) used for final elution of purified DNA (see Step 14) in a 65°C heat block or water bath to prewarm.)
   
   
3. Add 500μl of Nuclei Lysis/RNase Solution, mix by inversion and incubate at 80°C for 10 minutes. 
   
   
4. Prepare 1 Wizard^® SV Minicolumn assembly for each sample. Each Minicolumn assembly consists of a Wizard^® SV Minicolumn and a Collection Tube. Label the Collection Tube and place the Wizard^® SV Minicolumn assembly into a microcentrifuge rack.
   
   
5. Immediately transfer the entire contents of the bacterial cell lysate prepared in Step 3 into the  Wizard^® SV Minicolumn assembly. 
   
   
6. Centrifuge at 13,000 × g for 3 minutes to bind genomic DNA to the minicolumn. If any solution remains in the column, spin again for 1 minute.
   
   
7. Remove the Wizard^® SV Minicolumn from the assembly and discard the liquid in the Collection Tube. Replace the Wizard^® SV Minicolumn in the Collection Tube. 
   
   
8. Add 650μl of Wizard^® Wash Solution to each Wizard^® SV Minicolumn assembly.
   
   
9. Centrifuge at 13,000 × g for 1 minute.
   
   
10. Discard the liquid in the Collection Tube and replace the Wizard^® SV Minicolumn in the Collection Tube.
    
    
11. Repeat Steps 8–10 three times for a total of four wash steps.
    
    
12. After the last wash, empty the Collection Tube and reassemble the Wizard^® SV Minicolumn assembly. Centrifuge at 13,000 × g for 2 minutes to dry the binding matrix.
    
    
13. Remove the Wizard^® SV Minicolumn and place into a clean, labeled 1.5ml microcentrifuge tube for elution (not provided).
    
    
14. Add 200μl of prewarmed DNA Rehydration Solution or nuclease-free water (see Step 2) and incubate at room temperature for 2 minutes.
    
    
15. Centrifuge the Wizard^® SV Minicolumn/elution tube assembly at 13,000 × g for 1 minute.
    
    
16. Remove the Wizard^® SV Minicolumn and discard. Cap the elution tube containing the purified genomic DNA and store at –20°C to –70°C.

Isolation of Genomic DNA from Gram-negative Bacteria

Materials to Be Supplied By the User

• Wizard^® SV DNA Purification System (Cat.#A2360, A2361)
• 95% ethanol
• 80°C heat block or water bath
• 65°C heat block or water bath
• 37°C heat block or water bath
• 1.5ml microcentrifuge tubes
• microcentrifuge
• DNA Rehydration Solution (optional) (Cat.# A7963)

Preparation of Reagents

Prepare the  Wizard^® SV Genomic DNA System components as directed in the Wizard^® SV Genomic DNA Purification System Technical Bulletin #TB302.

Nuclei Lysis/RNase Solution: For each extraction, combine 500μl of Nuclei Lysis Solution and 7μl of RNase Solution (both are provided with the Wizard^® SV Genomic System).

Genomic DNA Purification Procedure: Gram-negative Bacteria

1. Pellet 0.5ml of bacterial culture by centrifugation (21,000 × g for 2 minutes). Discard supernatant. At this time, place the DNA Rehydration Solution (or nuclease-free water) used for final elution of purified DNA (see Step 13) in a 65°C heat block or water bath to prewarm.)
   
   
2. Resuspend the bacterial cell pellet with 500μl of Nuclei Lysis/RNase Solution. Incubate at 80°C for 10 minutes.
   
   
3. Prepare one Wizard^® SV Minicolumn assembly for each sample. Each Minicolumn assembly consists of a Wizard^® SV Minicolumn and a Collection Tube. Label the Collection Tube and place the Wizard^® SV Minicolumn assembly into a microcentrifuge rack.
   
   
4. Immediately transfer the entire contents of the bacterial cell lysate prepared in Step 2 into a Wizard^® SV Minicolumn assembly. 
   
   
5. Centrifuge at 13,000 × g for 3 minutes to bind genomic DNA to the Wizard^® SV Minicolumn. If some solution remains in the column, spin again for 1 minute.
   
   
6. Remove the Wizard^® SV Minicolumn from the minicolumn assembly and discard the liquid in the Collection Tube. Replace the Wizard^® SV Minicolumn in the Collection Tube.
   
   
7. Add 650μl of Wash Solution to each Wizard^® SV Minicolumn assembly.
   
   
8. Centrifuge 13,000 × g for 1 minute.
   
   
9. Discard the liquid in the Collection Tube and replace the Wizard^® SV Minicolumn in the empty Collection Tube.
   
   
10. Repeat steps 7–9 three times for a total of four wash steps.
    
    
11. After the last wash, empty the Collection Tube and reassemble the Wizard^® SV Minicolumn assembly. Centrifuge at 13,000 × g for 2 minutes to dry the binding matrix.
    
    
12. Remove the Wizard^® SV Minicolumn and place into a clean, labeled 1.5 ml microcentrifuge tube for elution (not provided).
    
    
13. Add 200μl of prewarmed DNA Rehydration Solution or nuclease-free water (see Step 1) and incubate at room temperature for 2 minutes.
    
    
14. Centrifuge the Wizard^® SV Minicolumn/elution tube assembly at 13,000 × g for 1 minute.
    
    
15. Remove the Wizard^® SV Minicolumn and discard. Cap the elution tube containing the purified genomic DNA and store at –20°C to –70°C.

Analysis of Purified Genomic DNA

Purified genomic DNA was eluted in 200μl of DNA Rehydration Solution (or nuclease-free water). Genomic DNA was isolated from four Gram-positive and four Gram-negative bacterial strains (Table 1) using two methods, a solution-based purification method using the Wizard^® Genomic DNA Purification Kit (Cat.# A1120, A1125; see the Wizard^® Genomic DNA Purification Kit Technical Manual #TM050 for details) and the Wizard^® SV Genomic DNA Purification System. Ten microliters of DNA isolated using each method was analyzed by agarose gel electrophoresis. The results are shown in Figure 1. Both purification methods yielded high molecular weight DNA with little degradation. For Gram-negative strains, yield from the solution-based purification method was higher than that obtained with the Wizard^® SV Genomic System (Figure 1B). However, the amount of DNA isolated using the  Wizard^® SV System was still more than enough for fbAFLP analysis.

DNA samples purified using the traditional solution-based method or the Wizard^® SV Genomic System were also compared in fbAFLP analysis. Figure 2 shows the results of fbAFLP analysis with Staphylococcus aureus and Salmonella Typhimurium DNA. Results obtained from DNA purified by the solution-based method are overlaid with those obtained using SV-purified DNA. Figure 2 shows that similar results were obtained in fbAFLP analysis, regardless of the DNA purification method used.

Conclusion

The Wizard^® SV Genomic DNA Purification System can be used to purify genomic DNA from a variety of both Gram-positive and Gram-negative bacteria. The purified genomic DNA is high quality and suitable for restriction/amplification applications such as fbAFLP.

The key advantage of the Wizard^® SV System is the absence of the intricate manipulations often associated with protein precipitation and alcohol precipitation of nucleic acids. Reproducible results also indicate  potential for high-volume extraction batches. Eventual increase in scale using the VacMan^® Vacuum Manifold (Cat.# A7231, A2291), the 96-well SV plate kit (Wizard^® SV 96 Genomic DNA Purification System; Cat.# A2370, A2371), and eventually “walk-away” robotic automation makes the SV System a good fit for the increasing pressures facing many clinical laboratories today.

References

1. Antonishyn, N.A., et al (2000) Evaluation of fluorescence-based amplified fragment length polymorphism analysis for molecular typing in hospital epidemiology: comparison with pulsed-field gel electrophoresis for typing strains of vancomycin-resistant Enterococcus faecium. J. Clin. Microbiol. 38:4058–4065.
2. Savelkoul, P.H.M., et al (1999) Amplified-Fragment Length Polymorphism analysis: the state of an art. J. Clin. Microbiol. 37:3083–3091.
3. Vos, P. et al (1995) AFLP: a new concept for DNA fingerprinting. Nucleic Acids Research 23:4407–4414.

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